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Metabolomics : Open Access
http://dx.doi.org/10.4172/2153-0769.1000158
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ISSN: 2153-0769 s

Research Article Open Access

Isolation and Characterization of Coumarin Isolated from Endophyte,

Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa
Cancer Cell Line
Umashankar T1, Govindappa M1*, Yarappa Lakshmikantha R2, Padmalatha Rai S3 and Channabasava1
1
Endophytic Natural Product Laboratory, Department of Biotechnology, Shridevi Institute of Engineering and Technology, Sira Road, Tumkur-572 106, Karnataka, India
2
Department of P.G. Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, JnanaSahyadri, Shankaraghatta, Shimoga, Karnataka -577 451, India
3
Department of Biotechnology, School of Life Sciences, Planetorium Complex, Manipal University, Manipal-576 104, Udupi Dist, Karnataka State, India

Abstract
Endophyte, Alternaria species-1 isolated from leaf part of Crotalaria pallida was selected in isolation, identification
and purification of coumarin. HPLC, UV-spectrophotometry, FTIR, NMR and XRD studies were carried out on
characterization of endophytic fungal coumarin. In vitro apoptotic activity was carried out against HeLa cervical cancer
cell lines. The results of MTT studies, acridine staining activity revealed the coumarin as an effective in inducing apoptotic
activity in HeLa cells. The coumarin significantly inhibited the proliferation of HeLa cells and its a concentration and
time dependent manner. Significant elevation of caspase 3 and 9 and activity was observed in coumarin treated HeLa
cells but no elevation or activity of caspase 7, 8, 10 was not observed. Coumarin has shown 0.156 g/ml of significant
IC50 value against viable of HeLa cell lines. Thus, coumarin exerts apoptotic activity by caspase dependent apoptosis
by enhancing the caspase 3 and 9 and it degraded the DNA (avoided the further replication). This is the first report in
internationally that coumarin isolated from endophyte, Alternaria species-1, purified and characterized and its role was
identified in apoptotic activity.

Keywords: Endophyte; Alternaria sp.; Coumarin; HPLC; XRD;
FTIR; Apoptosis
Introduction
Cancer is a generic term refers to the large group of diseases
characterized by abnormal growth of cells by rapidly beyond their
usual boundaries, invading to other parts of the body organs leading
to the formation of malignant tumors and neoplasmas. Cancer causes 1
in 8 deaths and rapidly becoming pandemic all over the globe (WHO,
2015). There were around 14.1 million new cancer cases, 8.2 million
cancer deaths and 32.6 million are living with cancer (IARC, 2012). This
scenario of cancer has made focused on searching of new effective and
economical both in usage and production of therapeutic agents. The
cancer treatment is prolonged one, it is necessary to reinvestigate the
therapeutics of short duration, less side effects, and economical. In this
context natural products are considered to be the choice as apoptotic
agents.
Coumarins are the benzopyrone compounds belong to flavonoid
groups of secondary metabolites of plants. Presently there are more
than 1300 coumarins have been identified in plants, bacteria, and
fungi [1-3]. Coumarins have their specific fingerprints as antiviral [4],
antimicrobial [5], antioxidant, anti-inflammatory [6,7], antiadipogenic
[8], cytotoxic [9], apoptosis [10], antiprolilferative [11], antitubercular
[12] and cytotoxicity [13] agent(s). Due to these wide range of
pharmacological values, coumarins and its derivatives has got more
importance in synthesis and production.
Crotalaria is one of the largest genera in tropical Africa. The genus
includes 690 species that are mainly situated in Africa and Madagascar
[14]. Species have also been found in India, United States of America
(USA) and China. Crotalaria pallida Aiton (Fabaceae) is an erect shrub,
annual or short-lived perennial herb of 1.5 m or more tall. The plant
has been used as traditional medicine; its roots have been used to treat
swelling of the joints and its leaves as vermifuge [15]. Coumarins were
having been reported from different species of Crotalaria [16,17]. In
this urge, the effective isolation of coumarins by the endophytes of
Crotalaria pallida was felt worthy. Endophytes are any ubiquitous
organisms, bacteria or fungi, occurring within plant tissues, distinct
from the epiphytes that live on plant surfaces [18]. They inherit the
characteristics of host plants in secreting the secondary metabolites
[19-21]. These inheritance properties of endophytes are beneficial
industrially in the production of important secondary metabolites.
The present study was aimed on, isolation and identification of
endophytes of Crotalaria pallida, purification and characterization of
endophytic coumarin by HPLC, UV spectra, XRD, FTIR and NMR,
in vitro apoptotic activities on HeLa cell lines by MTT assay, acridine
orange staining, analysis of caspase-3, 7, 8, 9 and 10 activity.
Materials and Method
Collection and mass production of leaf endophyte, Alternatia
species-1
Collected the leaf endophyte, Alternaria species-1 from stock
culture unit of Department of Biotechnology, Shridevi Institute of
Engineering & Technology, Tumkur, India were mass cultured on PDB
broth for large scale cultivation which was then incubated at room
temperature 26 2C for 8 days.
*Corresponding author: Govindappa M, Endophytic Natural Product Laboratory,
Department of Biotechnology, Shridevi Institute of Engineering and Technology,
Sira Road, Tumkur-572 106, Karnataka, India, Tel: +91-7203238327; Fax: +91-
816-2212628; E-mail: dravidateja07@gmail.com, endophytessiet@gmail.com
Received November 17, 2015; Accepted December 08, 2015; Published
December 10, 2015
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha
Rai S, Channabasava. (2015) Isolation and Characterization of Coumarin Isolated
from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action
on HeLa Cancer Cell Line. Metabolomics 5: 158. doi:10.4172/2153-0769.1000158
Copyright: 2015 Umashankar T, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

Metabolomics
ISSN: 2153-0769 JOM an open access journal Volume 5 Issue 4 1000158
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha Rai S, Channabasava. (2015) Isolation and Characterization
of Coumarin Isolated from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa Cancer Cell Line.
Metabolomics 5: 158. doi:10.4172/2153-0769.1000158
Microwave-assisted extraction (MAE)
After incubation, the fungal mycelium mat was taken for extraction
using ethanol. Based on the earlier report of Umashankar et al.
[22], Microwave Assisted Extraction (MAE) method was used, the
endophytic fungal mat mixed with ethanol was kept for extraction in
microwave method at 2 cycles of 5 minutes each at 100C.
Identification of coumarins in the extracts
Extract containing coumarins was confirmed by the confirmation
tests [23]. Extracts containing phenolic compound were confirmed by
the confirmation tests [24].
Test 1: 3 ml of ethanol extract was evaporated to dryness in a vessel
and the residue was dissolved in hot distilled water. It was then cooled
and divided into two test portions, one was reference, second was the
test. To the second test tube added 0.5 ml of 10% ammonium hydroxide.
The occurrence of intense fluorescence indicates the presence of
coumarins and derivatives [23].
Test 2: To the concentrated alcoholic extract, added few drop of
alcohol FeCl3 solution deep green colour formed which turned to
yellow on addition of conc. HNO3 indicates the presence of coumarins.
Test 3: The alcohol extract of drug was mixed with 1 N NaOH
solution (one ml each). Development of blue green fluorescence
indicates presence of coumarins.
Test 4
Detection of phenols: In beakers, 5 ml of each previous filtered
extract were taken and 1ml of FeCl3 (1%) and 1 ml K3(Fe(CN)6) (1%)
were added. The appearance of fresh radish blue color indicated the
presence of polyphenols [24]
Isolation, purification of coumarin from Alternaria species-1
from high performance liquid chromatography (HPLC)
Identification, isolation and purification of coumarin were done
using Alternaria species-1ethanol extract based on our earlier report
[13].
The obtained chromatograms were compared with standards based
on retention at 18.7 min is p-coumaric acid, 30.227 min is 2- hydroxyl
cinnamic acid and 31.231 min is coumarin.
UV-Visible spectrophotometry
The coumarin absorption was carried out on a UV
spectrophotometer at 305 nm in quartz cell. The coumarin was well
known for their absorbance in the UV region of the spectra because of
the mesomeric effect of the double bonds, and their phenolic cycle. The
polyphenols are detectable, then at low concentration (110 l M).
Fourier transform infrared spectroscopy (FTIR)
The powder material and Alternaria species-1 mycelia mass
was ground with a specially purified potassium bromide in a mortar
and pestle. The ground mixture was then pressed in a mechanical
press to form a translucent pellet. The infrared spectra of the
materials were recorded using Agilent (Cary-630) Fourier transform
spectrophotometer in the range of 400-4000 cm-1.
X-ray diffraction (XRD) studies
The synthesized sample is gently grounded in an agar mortar
and pestle. The fine powder is packed into a sample holder having a
Metabolomics
ISSN: 2153-0769 JOM an open access journal
Page 2 of 8
diameter of 1 cm and depth of 3 mm. The surface of the packed sample
is smoothed with a flat glass and then powder X-ray diffraction spectra
of the samples are recorded on Xpert Pro X-ray diffractometer. The
powder XRD involves the use of the collimated beam of X-rays; with
wave lengths Cu K radiation of wavelength in the range 0.5 to 2 ,
incident on a specimen, which is diffracted by the crystalline phase in
the specimen. The identification of the phase(s) of the as sample was
carried out by x-ray powder diffraction (PANalytical Xpert X-ray
powder diffractometer) using Cu-K radiation at =1.5418 . A scan
step of 1 s and a step size of 0.1 (in 2 ) were applied to record the
patterns in the range of 5 to 70 (2 ).
The Powder XRD pattern has been used to determine the symmetry
of the crystallographicunit cellof the coumarin.
C-13 NMR
The solid coumarin was tested by NMR. The NMR spectrum of the
crystalline coumarin was done in order to be compared to other spectra.
The NMR tests were recorded at 300 MHz on a Brucker spectrometer.
Chemical shifts are expressed in part per million.
In vitro apoptosis activity on HeLa cell lines
HeLa (cervical cancer) cell lines was obtained from NCCS (National
Centre for Cell Science), Pune. The HeLa cells were cultured in DMEM
10% FBS complete medium. The medium was supplemented with 10%
heat inactivated fetal bovine serum and antibiotics. The cell lines were
maintained at 37C in a 5% CO2 incubator and the media were changed
frequently.
MTT assay
This is a colorimetric assay that measures the reduction of yellow
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
by mitochondrial succinate dehydrogenase. The MTT enters the cells
and passes into the mitochondria where it is reduced to an insoluble,
coloured (dark purple) formazan product. The cells are then solubilised
with an organic solvent and the released, solubilised formazan reagent
is measured spectrophotometrically. Since reduction of MTT can only
occur in metabolically active cells, the level of activity is a measure of
the viability of the cells. Experimental designs were done at 24 and 48h
based on procedure of Umashankar et al. [13].
Detection of apoptosis by acridine orange staining
Detection of apoptosis in HeLa cell lines is characterized by the
double staining with acridine orange and ethidium bromide [25]. Live
cells are observed by bright dots and dead cells by orange to brown
colour. 2 104 cells per well were seeded in 96 well plate and treated
for 48 h. After incubation, the plates were centrifuged. 10 l of 1 mg/
ml Acridine orange and Ethidium bromide mixture was added to each
well. Nuclei were visualized and photographed under the fluorescent
microscope. The differentiation in the staining of live and dead cells was
observed with these two stains. Acridine orange stains both live and
dead cells. The ethidium bromide stains only the dead cells by became
organization due to intercalate DNA of cells with lost or degraded DNA
of cell integrity.
Determination of caspases activities
The cells were harvested and washed in PBS at 4C after incubation.
Using caspase apoptosis kit, the caspases 3, 7, 8, 9 and 10 enzymes
activities were analyzed and quantified [26,27] in coumarin treated
HeLa cell line. All measurements were performed 3 times, and changes
Volume 5 Issue 4 1000158
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha Rai S, Channabasava. (2015) Isolation and Characterization
of Coumarin Isolated from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa Cancer Cell Line.
Metabolomics 5: 158. doi:10.4172/2153-0769.1000158

Page 3 of 8

in absorbance of each caspases were measured at 405 nm using an Phytochemicals Alternaria species-1 (Leaf)
ELISA reader. Phenols +++
Ten g/ml of Alternaria species-1 coumarin treated the HeLa cells Coumarin test 1 +++
to induce apoptosis, cells treated with DMSO and phosphate buffered Coumarin test 2 +++
saline (pH 7.2) were used as control, standard drug 5-fluorouracil
Coumarin test 3 +++
(0.26 g) was used as positive control. After treatment, the cells were
incubated for 48 h. After incubation, the cells were collected and
*
Repeated the each experiment thrice, +=Presence, ++=Medium, +++=More and
-=Absence
suspended with cell lysis buffer, cells were incubated on ice for 10 min
Table 1: Identification of coumarins and phenol in leaf endophyte, Alternaria species-1.
and centrifuged at 10,000 rpm for 5 min and supernatant obtained was
treated with 4 ml of 4 mM pNA-conjugated substrates (DEVD-pNA) Coumarins Leaf extract
Alternaria species-1
(Leaf)
and incubated for 3.5 h at 37C. The released amount of pNA from each
treatment was measured at 405 nm using ELISA microplates reader. p-Coumaric acid + +
Based treated to untreated cells the absorbance of the relative caspase 2-Hydroxy coumaric acid - -
activity was calculated.
Coumarin + +
To study the inhibitory action of caspase-3, 7, 8, 9 and 10 in the
extract, the sample was pre-incubated with inhibitor in cell lysate Note: +: presence, -: absence
samples at room temperature (26 2C) for 10 min before adding Table 2: Presence of two different coumarins in plant extract and Alternaria species-1.
caspases 3, 7, 8, 9 and 10 substrate solution separately.
DAD1 A, Sig=275,4 Ref=off (C:\CHEM32_...ATA\3002-HPLC-0002\AGILENT1810 2014-10-18 13-59-25\AG180000003.D)

Results

30.206 - 2-Hydroxy cinnamic acid

mAU

18.715 - p-Coumaric acid

40

31.217 - Coumarin
The endophyte, Alternaria species-1 have shown the presence of 30

coumarin in all the tested methods and also confirms the presence of 20

polyphenols, it indicates coumarin presence (Table 1). These methods

are commonly using to study coumarin presence in the extract.
10

Identification of coumarins in the extracts by HPLC -10

0 5 10 15 20 25 30 35 40 45 min

When we compared with standard coumarins, the results confirmed

Figure 1: Standard chromatogram of all the three coumarins in HPLC.
the presence of two coumarins (p-coumaric acid and coumarin) at high
per cent in ethanol extracts of Alternaria species-1 in HPLC method
(Table 2). Figure 1 depicts the standard coumarins in HPLC. The ethanol
extract of leaf part of Crotalaria pallida has shown three different types
DAD1 A, Sig=275,4 Ref=off (C:\CHEM32_...ATA\3002-HPLC-0002\AGILENT1810 2014-10-18 13-59-25\AG180000013.D)
mAU
2.684

of coumarins (Figure 2). The ethanol extract of fungal endophyte, 200

Alternaria species-1 yielded only two coumarins viz., p-coumaric acid

2.370
3.071

150

and coumarin, they were identified based on their retention time and
2.276

compared with standard in HPLC (Figure 3 and 4). Purified coumarin

18.741 - p-Coumaric acid

100
2.547

31.242 - Coumarin

based on retention time (31.514) and obtained single peak in HPLC

4.009

50
31.814

49.429
35.121
26.944
26.368
4.459

6.417

16.840

20.626

24.454

37.784
21.947
4.804
5.075

after collecting the bulk quantity (Figure 5). The coumarin was
9.637
7.478

9.241

isolated and purified from Alternaria species-1 using HPLC based on 0 5 10 15 20 25 30 35 40 45 min

retention time (31.514) and done the experiment repeatedly to collect Figure 2: HPLC chromatogram of leaf extract of Crotalaria pallida.
the purified coumarin at large quantity and run the same sample in
the HPLC to know its purity and to obtain pure coumarin for in vitro
apoptosis activity. The purified coumarin was characterized by other
specific analytical methods and parameters such as UV-Vis, XRD, FTIR mAU
DAD1 A, Sig=275,4 Ref=off (C:\CHEM32_...ATA\3002-HPLC-0002\AGILENT1810 2014-10-18 13-59-25\AG180000014.D)
2.550 2.386
2.688
3.069

5.498

and NMR studies. The results of these studies were very significant for
26.882

40

purity and confirmed. These methods were used to characterize and

4.478
4.018

30

confirm the coumarins isolated different sources [28-30].

18.744 - p-Coumaric acid

20
31.223 - Coumarin

Using diode array detector plotted the chromatographic/

49.373
38.500
11.631

10
3.521

35.110

spectrometric data images. It was used to facilitate HPLC method to

16.678

optimize and validity for detection conditions in off-line also (Figure -10
0 5 10 15 20 25 30 35 40 45 min

9). Based on linearity, sensitivity, precision, repeatability, and accuracy

the method was validated. The method was used to determine seven
phenolic compounds include p-coumaric acid [31,32]. To improve
the quantitative analysis method established the iso-plot method
and it is effective also. Using C18 column with gradient elution of
phosphoric acid aqueous solution (0.05%, v/v) and acetonitrile the
chromatographic separation was done, and adopted detection of
isolated p-coumaric acid using wave lengths switch programme and
optimized with iso-plot. Isolation and purification of coumarins were
carried out by the repeated performance and collection of specific
Figure 3: HPLC chromatogram of leaf Alternaria species-1 ethanol extract.
coumarins at their respective retention time. The purified coumarins
were again characterized by other specific analytical methods and
parameters. The purity of coumarin was confirmed by the isoplot,
3D plot, spectral studies, peak purity information, HPLC RT and 3D
plot of RT studies. The results of these studies were very significant.
Temperature (T) on one axis, pressure (p) on a second axis, and specific

Metabolomics
ISSN: 2153-0769 JOM an open access journal Volume 5 Issue 4 1000158
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha Rai S, Channabasava. (2015) Isolation and Characterization
of Coumarin Isolated from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa Cancer Cell Line.
Metabolomics 5: 158. doi:10.4172/2153-0769.1000158

Page 4 of 8

volume (v) on a third was done to coumarin using 3D plot graph. Based
these studies coumarin was isolated and purified and again subjected mAU
DAD1 A, Sig=275,4 Ref=off (C:\CHEM32_...ATA\3002-HPLC-0002\AGILENT1810 2014-10-18 13-59-25\AG180000021.D)

31.180 - Coumarin
HPLC to know the purity. Phenolic compounds absorb ultraviolet 20

region strongly and detection and quantification the spectra was used. 15

Detected the phenolic compounds by studying absorbance at 280 nm 10

from the eluent after separation with selecting mobile and stationary 5

phases. Our results confirmed by the results of Hosler et al. [24]. 0

The coumarin absorption was carried out on a UV 10 15 20 25 30 35 40 45 min

spectrophotometer at 305 nm in quartz cell. The coumarin was well Figure 5: Purified coumarin from Alternaria species-1 showing 31.130
known for their absorbance in the UV region of the spectra because of retention time.
the mesomeric effect of the double bonds, and their phenolic cycle. In
this range, studied absorption showed four absorption bands at 241 nm,
297 nm, 277 nm and 307 nm for the coumarin standard and coumarin
from the extract of Alternaria species-1 (Figure 6 and 7).
FTIR
The IR spectrum of coumarin shows lactone carbonyl at 1715 cm-1,
C=C at 1608 cm-1, 1450 cm-1 and C-O-C at 1254 cm-1. Infrared
spectroscopy involves the absorption of electromagnetic radiation in
the infrared region of the spectrum which results in changes in the
vibrational energy of molecules. Usually all molecules have vibrations
in the form of stretching, bending, etc., the absorbed energy will be
utilised in changing the energy levels associated with them [33]. The
IR spectrum of coumarin shows a sharp band at 3381 cm-1 associated
to the stretching -OH vibration. The band at 2963 cm-1 associated with
the aromatic CH-stretching. The band present at 1715 reflecting the
CO-stretching of carbonyl group of COOH (Figure 8 and 9) and it was
compared with standard and the purity of the compound is similar to
standard coumarin. The shows the FTIR spectrum of coumarin laser
dye and compared with chemical formula of this dye. There were more
than one peak obtained in region of the C-H bending vibrations out of
plane (900-600) cm-1 can support the presence of an aromatic structure. Figure 6: UV-visible spectra: coumarin from the extract of Alternaria species-1.
In region (1200-1000) cm-1, there is a peak at 1028.09 cm-1 refers to C-H
bending vibrations in of planes. The benzene rings very clear which
is supportive to the peak at 1489.10 cm-1 that acts the C=C stretching
(1400-1600) cm-1 of the benzene ring and a peak at 3061.13 cm-1 for
C-H stretching (3000-3100) cm-1.
XRD
The powder XRD pattern of the coumarin corresponds to

DAD1 A, Sig=275,4 Ref=off (C:\CHEM32_...ATA\3002-HPLC-0002\AGILENT1810 2014-10-18 13-59-25\AG180000012.D)

mAU
2.395
2.679

18.099 - p-Coumaric acid

31.514 - Coumarin

400

300
3.109
2.545

3.983

200
13.266

16.639

19.331

100

-100

0 5 10 15 20 25 30 35 40 45 min

Figure 7: UV-visible spectra: coumarin standard.

orthorhombic phase with space group Pcaz (Z=4) (Figure 10).

A typical analysis of a 13C NMR spectrum consists of matching
expected chemical shifts to the expected moieties. The chemical shifts
Figure 4: Separation of p-coumaric acid and coumarin from the crude were matched with the existed earlier reports. For coumarin, chemical
extract and its area summary. shifts 103.326, 109.818 and 103.322 were characteristic (Figure 6). The

Metabolomics
ISSN: 2153-0769 JOM an open access journal Volume 5 Issue 4 1000158
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha Rai S, Channabasava. (2015) Isolation and Characterization
of Coumarin Isolated from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa Cancer Cell Line.
Metabolomics 5: 158. doi:10.4172/2153-0769.1000158
Figure 8: FTIR spectra of standard coumarin.
Figure 9: FTIR spectra of purified coumarin from the extract of Alternaria species-1.
results are compared with IR spectra of standard coumarin (Figure 11).
In vitro apoptotic activity on HeLa cell lines
In vitro apoptotic activity on HeLa cell lines shows that, the
endophytic fungal coumarin from Alternaria species-1 has remarkable
apoptotic activity on cancer cell lines. MTT assay showed that the
decrease in the cell viability after 24 and 48 h. Apoptotic activity
of endophytic fungal coumarin was demonstrated by its inhibition
of proliferation of HeLa (cervical cancer) cells in a time and dose-
dependent manner. 0.156 g/ml of significant IC50 value was noticed
(Figure 11 and 12) (Table 3).
Acridine orange and ethidium bromide staining
Detection of apoptosis in HeLa cell lines stained with acridine
orange and ethidium bromide treated by coumarin has given significant
results. The live cells were appeared as bright green spots and dead cells
are as orange to brown. Control treated cells showed most of the live
cells and there are more dead cells at 48 h of treatment than the cells
treated for 24 h. Staining with orange or brown is due to the ethidium
bromide, which acts as intercalate agent with apoptic cell DNA (Figure
13 and 14).
Caspases are known to be involved in pivotal regulator of cellular
apoptosis [33]. We have identified the effect or role of coumarin on
activation of caspases 3, 7, 8, 9 and 10 by calorimetric method. The
p-coumaric acid effectively induced the activity of caspases 3 and 9 in
HeLa cells and no effect was observed in caspases 7, 8 and 10 activities.
Metabolomics
ISSN: 2153-0769 JOM an open access journal
Page 5 of 8
Figure 10: XRD patterns of the coumarin corresponds to orthorhombic phase.
Figure 11: C-NMR pattern of the coumarin from the extract of Alternaria species-1.
From the results, conclude that the coumarin isolated from Alternaria
species-1 treatment enhanced the cell death of HeLa cancer cell line by
activating caspases enzymes in intrinsic pathways (Figure 15).
Alternaira species-1 coumarin induced apoptosis in HeLa cells
was associated with typical apoptosis by nuclear DNA degradation and
induction of caspase-3 and 9 activity.
Discussion
Production of secondary metabolites from endophytes is a wide
area. It depends on the host plant, endophytic fungi, laboratory
conditions and other optimization parameters. Based upon our earlier
in vitro biological and HPLC screening studies [13,22], Alternaria
sp. have been selected for isolation and purification of coumarin and
its cytotoxicity on HeLa cell lines. Investigations of Taxol, the first
apoptotic agent recovered by an endophytic fungi Taxomyces andreanae
of Taxus brevifolia [34] and other apoptotic drugs, Paclitaxel from
Alternaria sp. of Taxus cuspidata, Ginkgo biloba and Taxus chinensis
[35-37], Podophyllotoxin from Alternaria sp. of Sinopodophyllum
hexandrum, Sabina vulgaris and Sinopodophyllum hexandrum [38-
40] strengthens this apoptotic study in relation to endophytes and the
selected Alternaria species.
The detailed analytical characterization studies of coumarin
confirmed its presence in endophyte Alternaria species-1 of Crotalaria
Volume 5 Issue 4 1000158
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha Rai S, Channabasava. (2015) Isolation and Characterization
of Coumarin Isolated from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa Cancer Cell Line.
Metabolomics 5: 158. doi:10.4172/2153-0769.1000158

Page 6 of 8

Figure 13: Detection of apoptosis in HeLa cell lines stained by acridine

orange treated by endophytic coumarin, A) Control, B) HeLa cell lines
after 24 h and C) HeLa cell lines after 48 h

Figure 12: In vitro apoptotic activity of endophytic coumarin from

Alternaria species-1 on HeLa cancer cell lines.

Drug
% of cell
concentration % of viability
death
(g)
24h 48h 24h 48h

0.078 88.664 0.01 87.38 0.02 11.34 0.02 12.62 0.01

0.156 48.36 0.02 39.9 0.02 51.64 0.02 60.1 0.01

0.312 17.784 0.01 14.03 0.01 82.21 0.01 85.7 0.02

0.625 8.684 0.01 9.303 0.01 91.31 0.02 90.97 0.02

1.25 1.352 0.01 1.143 0.02 98.64 0.01 98.87 0.01 Figure 14: Detection of apoptosis HeLa cell lines.
2.5 0.416 0.02 0.467 0.01 99.58 0.03 99.53 0.03

5 1.456 0.03 1.61 0.03 98.54 0.02 98.39 0.02

Vehicle control 97.8 0.02 97.5 0.02 2.2 0.0 2.5 0.0

Doxorubicin 10.13 0.1 07.0 0.1 89.87 0.2 93 0.1

IC50 0.156 g

Table 3: In vitro apoptotic activity of endophytic coumarin from Alternaria species

on HeLa cancer cell lines.

pallida. HPLC results were found similar with the results of standard
coumarin. FTIR results of endophytic coumarin were confirmed by the
earlier studies reported by Murthi et al. [41]. The XRD results were also
significant and confirmed by the reports of Ramaswamy [42]. Similar
works were carried out in characterization of coumarin in Justicia Figure 15: Changes in different caspase activity in HeLa cell line after
treatment with Alternaria species-1 coumarin.
pectoralis leaf [43], Melilotus suaveolens [44]. A cytotoxic activity of
endophytic coumarin of Alternaria species-1 on cancerous HeLa cell
lines was extensively significant having an IC50 value of 0.156 g/ml. protease, catalyzing the target specific cleavage of many key cellular
MTT assay, acridine orange staining and caspase-3 colorimetric studies proteins [47]. Caspase -7 is executioner caspases, activates the intrinsic
reveals that, the cyotoxicity of endophytic coumarin on viable of HeLa apoptotic pathways through cleavage of BID to induce efficient cell
cells is due to DNA degradation and induction of apoptic caspase-3 and 9 death, apoptotic cell development. Caspase-9 initiates apoptosis by
enzyme activity. Caspases trigger apoptosis through a well-orchestrated cleaving and thereby activating executioner caspases, mitochondrial
cascade [45,46]. Caspases are crucial mediators of programmed cell morphological changes, ROS production by cleaving and activating
death (apoptosis). Among them caspase-3 is frequently associated

Metabolomics
ISSN: 2153-0769 JOM an open access journal Volume 5 Issue 4 1000158
Citation: Umashankar T, Govindappa M, Yarappa Lakshmikantha R, Padmalatha Rai S, Channabasava. (2015) Isolation and Characterization
of Coumarin Isolated from Endophyte, Alternaria Species -1 of Crotalaria pallida and Its Apoptotic Action on HeLa Cancer Cell Line.
Metabolomics 5: 158. doi:10.4172/2153-0769.1000158
BID into tBID [48]. Caspase-10 is initiate caspase in death receptor
signaling [49].
The acridine orange fluoroscent study clearly reveals that the
cytotoxicity on HeLa cells is due to cytostasis but not through cell
necrosis. Apoptic activities of endophytic coumarin were rectified by
the recent works carried out in HeLa cells with different apoptotic
drugs and fungal secondary metabolites [50-52]. The natural coumarins
induces apoptosis like cell death in HeLa cells mediated by the release of
apoptosis inducing factor [53] but our source is different i.e. endophytic
fungi. Interestingly, we also found caspase depended apoptosis from
our experiment whereas, we found elevation of caspase-3 activity.
The coumarin induces morphological changes, G0/G1 arrest, DNA
fragmentation and increased the sub-G1 group, affected the production
of reactive oxygen species and Ca2+ concentration, depolarization of
mitochondrial membrane potential, the anti-apoptotic proteins Bcl-2
and Bcl-xL, and increased the expression of the pro-apoptotic protein
Bax, decreased the mitochondrial membrane potential and promoted
the release of cytochrome c and the activation of caspase-3 before
leading to apoptosis in human cervical cancer cells (HeLa) [54]. Our
endophytic fungal coumarin have shows some of the above results such
as caspase-3 activity, toxicity and DNA fragmentation in HeLa cancer
cells.
Conclusion
In conclusion, the endophytic coumarin was isolated, identified
by the detailed UV-Vis, HPLC, FTIR, XRD, NMR studies. Apoptotic
activity was confirmed by MTT assay, acridine orange staining and
more activity of caspase 3 and 9. The mechanism of endophytic
coumarin was assumed through DNA degradation and induction of
caspase-3 and 9 activities without any cell necrosis. Further, optimal
studies are needed to investigate in vivo condition, in dose fixation
and other etiological studies. This is the first report on identified the
coumarin produce endophyte, Alternaria species-1, its characterization
and apoptotic activity in nationally and internationally. The endophytic
fungi can be used for production of apoptotic agent, coumarin.
Acknowledgement
We thank Dr. MR Hulinaykar, Managing Trustee, Sri Shridevi Charitable
Trust (R.) and Dr. Nalini N, Principal, SIET, Tumkur, India for encouragement
and suggestions during the research, Dr. KG Bhat, Taxonomist, Udupi, for his
assistance in collection and identification of the plant. We thank the Spectroscopic
unit of Indian Institute of Science, Bangalore, Karnataka, India, for their extended
service in assisting the technical help during this research work.
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